BACKGROUND: Multipotent mesenchymal stromal cells (MSC) are promising candidates in the field of regenerative medicine and in several studies have been genetically modified to bring a new property to or enhance an existing one in these cells. Furthermore, MSC have been used as gene delivery vehicles. The success of these experiments depends on selecting an appropriate method for gene delivery to the cells. METHODS: MSC were isolated from rat bone marrow; their authenticity was checked by differentiation experiments as well as staining for cell-surface markers. A systematic approach was used to optimize five cationic polymer-based gene delivery methods (Lipofectamin2000, Effecten, Superfect, Polyfect and FuGENE HD). The transfection yield and cell viability of each method was measured after 48 h in three to six separate experiments with nine to 12 different ratios and amounts of DNA/transfection reagent. RESULTS: The isolated MSC were successfully differentiated to osteoblasts, adipocytes and chondroblasts. They were positive for rat CD90 and CD73 and negative for CD31, CD45, CD11b and VEGFR2 markers. The average transfection rates with optimum conditions were 5.18+/-2.72 (FuGENE HD), 8.72+/-4.52 (Effecten), 9.59+/-3.12 (Superfect), 16.29+/-7.44 (Polyfect) and 19.60+/-3.12 (Lipofectamine 2000). The toxicity was below 20% for all reagents. DISCUSSION: Moderate levels of transfection and acceptable cell viability could be achieved using Lipofectamine 2000 and Polyfect in optimized conditions. The results could be improved by gating and sorting live cells using a simple FSC-SSC gating.
BACKGROUND: Multipotent mesenchymal stromal cells (MSC) are promising candidates in the field of regenerative medicine and in several studies have been genetically modified to bring a new property to or enhance an existing one in these cells. Furthermore, MSC have been used as gene delivery vehicles. The success of these experiments depends on selecting an appropriate method for gene delivery to the cells. METHODS: MSC were isolated from rat bone marrow; their authenticity was checked by differentiation experiments as well as staining for cell-surface markers. A systematic approach was used to optimize five cationic polymer-based gene delivery methods (Lipofectamin2000, Effecten, Superfect, Polyfect and FuGENE HD). The transfection yield and cell viability of each method was measured after 48 h in three to six separate experiments with nine to 12 different ratios and amounts of DNA/transfection reagent. RESULTS: The isolated MSC were successfully differentiated to osteoblasts, adipocytes and chondroblasts. They were positive for ratCD90 and CD73 and negative for CD31, CD45, CD11b and VEGFR2 markers. The average transfection rates with optimum conditions were 5.18+/-2.72 (FuGENE HD), 8.72+/-4.52 (Effecten), 9.59+/-3.12 (Superfect), 16.29+/-7.44 (Polyfect) and 19.60+/-3.12 (Lipofectamine 2000). The toxicity was below 20% for all reagents. DISCUSSION: Moderate levels of transfection and acceptable cell viability could be achieved using Lipofectamine 2000 and Polyfect in optimized conditions. The results could be improved by gating and sorting live cells using a simple FSC-SSC gating.
Authors: Nupura S Bhise; Ryan S Gray; Joel C Sunshine; Soe Htet; Andrew J Ewald; Jordan J Green Journal: Biomaterials Date: 2010-07-31 Impact factor: 12.479
Authors: Joana S Boura; Francisco Dos Santos; Jeffrey M Gimble; Carla M P Cardoso; Catarina Madeira; Joaquim M S Cabral; Cláudia Lobato da Silva Journal: Hum Gene Ther Methods Date: 2013-02 Impact factor: 2.396
Authors: Margaret A McNulty; Amarjit S Virdi; Kent W Christopherson; Kotaro Sena; Robin R Frank; Dale R Sumner Journal: Clin Orthop Relat Res Date: 2012-09 Impact factor: 4.176